Descriptions

Intracellular dNTP pool sizes are highly asymmetric, with dGTP usually
comprising 5 to 10% of the sum of the dNTP pools. The work presented in this
dissertation addresses the question of whether the underrepresentation of dGTP is
related to its potential to be oxidized by reactive oxygen species. 8-oxo-guanine is
important in oxidative mutagenesis, and current evidence indicates that this lesion
arises in DNA partly through oxidation of dGTP, followed by incorporation of 8-oxo-dGTP
into DNA. The bacterial MutT protein and its mammalian homolog catalyze the
hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP in vitro. It is a widely accepted premise
that the primary function of these enzymes is to remove 8-oxo-dGTP from the
nucleotide pool of cells so that it cannot be used as a substrate for DNA synthesis.
However, this model has been called into question by observations that some mutT
strains of E. coli display a mutator phenotype when grown anaerobically, and by
kinetic studies that showed 8-oxo-dGTP to be a poor DNA polymerase substrate.
In this study, the dNTP pools of mammalian cells cultured in varying oxygen
conditions were measured, with the expectation that the dGTP pool would expand
under low oxygen conditions if it were a target for damage by reactive oxygen species.
HeLa cells cultured in 2% 0₂ showed no change in the dGTP pool when compared to
cells cultured in 20% 0₂; however, in V79 cells, the dGTP pool did expand in 2% 0₂.
This result was not specific to the dGTP pool, as pools of dATP and dTTP also
increased when V79 cells were cultured at 2% 0₂. These results suggest that there may
be increased turnover of the dGTP pool when cells are cultured in high oxygen, but
these experiments did not address the reason for this oxygen-dependent change.
In order to determine whether 8-oxo-dGTP accumulates to levels that are
sufficient to cause mutagenesis in cells, an analytical method for the measurement of
8-oxo-dGTP from cell extracts was developed. By use of this method, which involves
reversed-phase high performance liquid chromatography coupled with electrochemical
detection, no 8-oxo-dGTP was detected in mutT E. coli cells, even when they were
cultured in the presence of H₂0₂. The estimated upper limit of 8-oxo-dGTP in these
cells is about 240 molecules per cell, which corresponds to an intracellular
concentration of approximately 0.34 μM. When 8-oxo-dGTP was added at this
concentration to an in vitro DNA replication system in which replication errors could
be scored as mutations, along with the four normal dNTPs at their estimated
intracellular concentrations, there was no detectable effect on the frequency of
mutation. Therefore, the presence of 8-oxo-dGTP at physiologically relevant
concentrations does not appear to be significantly mutagenic. The results presented in
this dissertation suggest that the mechanism by which the MutT enzyme counteracts
mutagenesis should be reevaluated.